Development and Validation of a Novel In Vitro Model for the Assessment of Heterocellular Interactions Mediated by Connexin43

The injury border zone (IBZ), a region of transitional tissue between intact myocardium and the ischemic area, is often the site of lethal reentrant arrhythmia generation in post-myocardial infarction (MI) patients. Disruption to normal connexin43 (Cx43) localization at the intercalated disc (ID), separation of myocytes by activated fibroblasts and deposited scar tissue are thought to be factors that render the IBZ a pro-arrhythmic substrate, though there is a current need to better understand these changes so directed therapies can be developed. There are no clinically available therapies focused on the mechanistic changes in the IBZ. Additionally, generation of new compounds is challenged by the inefficiencies of the drug development pipeline, including the use of in vitro testing platforms that are of little relevance to human disease. In order to address the need for directed therapies and improved testing platforms, we developed a 3D in vitro aggregate model of the IBZ that was validated against a murine cryo-injury model of infarction. This model was used to examine the changes in heterocellular myocyte-fibroblast interactions via Cx43 and cell organization patterns in response to αCT1 – a Cx43 carboxyl-terminal mimetic peptide which was previously shown to alter cell organization in the IBZ of a murine cryo-injury model. Imaging results indicated that αCT1 increased fibroblast migration, as well as myocyte cohesion, as evidenced by increased cell clustering in aggregates at 72 hours. This was likely an effect of Cx43 adhesion properties. Optical mapping studies indicated that αCT1 could protect cryo-injured hearts against conduction delay through the IBZ in conditions of altered extracellular ion concentration. Subsequent analyses from a gap junction (GJ) intercellular communication (GJIC) assay indicated that this may be a result of altered communication, as αCT1 was found to decrease GJIC in myocyte-fibroblast connections and in fibroblast-fibroblast connections. This decrease in GJIC was correlated with an increase in Cx43 phosphorylated at serine 368 (pS368) in myocyte-fibroblast interactions in vitro an in vivo, which is known to decrease GJ channel conductance. Super-resolution imaging of GJ plaques revealed a novel sub-domain organization with respect to non-phosphorylated Cx43 and Cx43 pS368. Together, the data provided in this dissertation describe the development of a novel in vitro model of the IBZ of relevance to drug response and disease mechanism that can be used as a high-throughput testing platform for candidate compounds. These studies, furthermore, suggest the importance of Cx43 heterocellular interactions as a therapeutic target for reducing cardiac arrhythmias